ARL3 GTPases facilitate ODA16 unloading from IFT in motile cilia

Eukaryotic cilia and flagella are essential for cell motility and sensory functions. Their biogenesis and maintenance rely on the intraflagellar transport (IFT). Several cargo adapters have been identified to aid IFT cargo transport, but how ciliary cargos are discharged from the IFT remains largely unknown. During our explorations of small GTPases ARL13 and ARL3 in Trypanosoma brucei, we found that ODA16, a known IFT cargo adapter present exclusively in motile cilia, is a specific effector of ARL3. In the cilia, active ARL3 GTPases bind to ODA16 and dissociate ODA16 from the IFT complex. Depletion of ARL3 GTPases stabilizes ODA16 interaction with the IFT, leading to ODA16 accumulation in cilia and defects in axonemal assembly. The interactions between human ODA16 homolog HsDAW1 and ARL GTPases are conserved, and these interactions are altered in HsDAW1 disease variants. These findings revealed a conserved function of ARL GTPases in IFT transport of motile ciliary components, and a mechanism of cargo unloading from the IFT.

The PDF file includes: Figs. S1 to S7 Table S1 Legends for movies S1 to S3 Other Supplementary Material for this manuscript includes the following:

Fig. S1 .
Fig. S1.TbARL3A and TbARL3C are synthetically lethal.(A) T. brucei cells were stably transfected with tetracycline (Tet)-inducible RNAi of TbARL3A and TbARL3C, individually or together.In control cells, the flagellum is laterally attached to the

Fig. S3 .
Fig. S3.The axonemal association of TbIC1 is partially reduced upon TbODA16 RNAi.(A and C) TbODA16 RNAi was induced in cells expressing mNG-Ty fusion to TbIC2 (A), TbIC1 (C).Scale bars: 10 μm.Their flagellar intensity was measured along the distal 1.5 µm and shown in (B) and (D), respectively.The results were shown as mean ± SD.P values were calculated by unpaired t test with Welch's correction.

Fig. S4 .
Fig. S4.Depletion of TbARL3C but not TbARL3A affects TbODA16 distribution.(A) Immunofluorescence of cells with endogenously expressed TbODA16-mCherry, TbARL3A-mNG-Ty and 3×HA-TbARL3C upon induction for TbARL3A RNAi or TbARL3C RNAi for 72 hours.Scale bar: 10 μm.(B) Comparison of TbODA16 intensity at the ciliary tip before and after RNAi of specified TbARL3 GTPases.The intensity was measured along the distal 1.5 µm of the flagellum.The results were shown as mean ± SD.P values were obtained from one-way ANOVA with Tukey's multiple comparisons test.(C) Immunoblots confirming efficient and specific TbARL3 depletion in each of the RNAi cell lines shown in this figure as well as in Fig. 4A.

Fig. S5 .
Fig. S5.Dual silencing of TbARL3A and TbARL3C stabilizes TbODA16-IFT interaction.(A-F) Cells stably expressing HA-tagged TbODA16 and YFP-tagged IFT subunits IFT46 (A-C) or IFT81 (D-F) were induced for TbARL3A/TbARL3C dual RNAi or not.TbODA16-IFT interaction was assessed by co-IP using GFP-Trap (A and D) or anti-HA affinity gel (B and E).Immunofluorescence showing TbODA16 in control and TbARL3A/TbARL3C dual RNAi cells (C and F).Scale bars: 10 μm.(G) Immunoblots confirming the depletion of TbARL3A and TbARL3C in all cell lines used in experiment shown in this figure and in Fig. 5.

Fig. S7 .
Fig. S7.TbODA16 is associated with TRiC.(A) Immunoblots probed with streptavidin-HRP revealed the biotinylation profiles of TbODA16-BioID2.The cells were extracted with 1% NP40 in PEM buffer and centrifuged to obtain whole cell (E), detergent soluble (S) and detergent insoluble (P) fractions.WT, wild type cells not expressing TbODA16-BioID2 fusion; no RNAi, cells with expression of TbODA16-BioID2 but not induced for RNAi; TbARL3A+TbARL3C dual RNAi 48 hours, cells with expression of TbODA16-BioID2 and induced for TbARL3A/TbARL3C dual RNAi for 48 hours.* ~75kDa bands corresponding to the size of TbODA16-BioID2.** ~60 kDa bands corresponding to the size of TRiC subunits.(B) Co-IP assays confirming the interaction between TbODA16 and TRiC subunit TCP-1-zeta.Cells expressing GFP were used as a negative control.